PEBBLEs are the smallest biosensors ever developed

EDITORS: Black-and-white photograph is available on request.

ANN ARBOR—University of Michigan researchers have found a way to monitor changes in the biochemistry of living cells by shooting the cells full of PEBBLEs.
Made of polymers, instead of stone, PEBBLEs (Probes Encapsulated By BioListic Embedding) are the smallest biosensors ever developed, according to Raoul Kopelman, the U-M's Kasimir Fajans Professor of Chemistry, Physics and Applied Physics. They were designed to work inside mammalian cells where they can detect subtle changes in concentrations of ions and small molecules.

"PEBBLEs are self-contained sensors powerful enough to detect even slight changes in cell biochemistry, but small enough to avoid damaging the cell," said Heather Clark, a U-M graduate student in chemistry. Clark is part of a U-M team currently developing and testing the biosensing devices in a research project funded by the U.S. Defense Advanced Research Projects Agency (DARPA).

Clark will present results from her research this week in a presentation at PITTCON '98, the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, taking place in New Orleans.

"For the first time, we can observe real-time chemical processes inside a living cell," said Kopelman. "The goal of the DARPA project is to learn what happens inside the cell when it is exposed to neurotoxic agents. If we can learn exactly how these toxins trigger a flood of ions in and out of cells, we may be able to speed up development of antidotes or countermeasures for lethal biological warfare agents."

Kopelman added that the ability to "watch what's happening inside a cell" has a wide variety of potential applications in other fields—including cancer therapy, glucose monitoring, drug or chemical toxicity testing, and all areas of biosensing.

Kopelman says Clark has fabricated PEBBLEs as small as 20 nanometers in diameter. That's 100,000 times smaller than the letter "n" in this sentence. The tiny polymer spheres contain many surface pores. When Clark adds dyes to a polymer microemulsion suspension during pebble fabrication, the dyes are naturally taken up by the PEBBLE's pores.

According to Clark, each dye is specific for, or will bind to, just one type of ion or molecule. So far, she has produced PEBBLEs specific for calcium, oxygen, magnesium and pH—the acidity level in a solution. When a PEBBLE is exposed to even very small quantities of its target substance, the dye in the PEBBLE glows when activated by a specific wavelength of light. As the concentration of the targeted substance changes, the intensity of the PEBBLE's fluorescence increases or decreases.

Once the PEBBLEs are ready, Clark uses pico-injection techniques or a gene gun to fire them randomly into human or mouse cells in a culture dish. "The PEBBLEs blast through the cell membrane like bullets," Clark explains, "but because they are so small, they rarely do any damage. Mortality of cells shot with PEBBLEs is only 2 percent higher than in control cells."

Martin A. Philbert, U-M assistant professor of toxicology in the School of Public Health, has directed toxicology tests on several types of PEBBLE-containing cells and agrees that PEBBLEs can be inserted with far less trauma to the cell than is produced by free dyes or fiberoptic probes. Philbert and Marion Hoyer, a U-M research fellow, will present results from these toxicology tests this week at the Society of Toxicology annual meeting in Seattle, Wash.

Others currently involved in the research project include Brian Athey, assistant professor of anatomy and cell biology in the U-M Medical School; U-M graduate students Alex Ade, Murphy Brasuel and Michael Miller; and U-M research fellows Steve Parus and Zhong-You Shi. The U-M has applied for a patent on its PEBBLE technology.